During cellular differentiation, new genes are turned on and new organelles appear. The appearance of new organelles with unique protein compositions suggests that differentiation requires the induction of new sorting machinery. We will address the question of de novo induction of organelles by a study of synaptic vesicle biogenesis. The synaptic vesicle, an easily purified organelle of unique size and composition, appears in large numbers in the nerve terminal during synaptogenesis and neuronal plasticity. We will ask if sorting machinery is required to selectively target membrane proteins to synaptic vesicles and if it too is tissue-specific and developmentally regulated. During phase I, we will identify sequences present on synaptic vesicle proteins that are necessary and sufficient for sorting to synaptic vesicles and identify candidate "sortases" that recognize the sorting domains. To identify sorting domains, fusion chimeras formed between membrane proteins selectively targeted to synaptic vesicles and those that are not will be stably expressed in PC12 cells (a neuroendocrine cell line that makes synaptic vesicles) and assayed for targeting to synaptic vesicles. Candidate "sortases" will be identified by two assays: 1) binding of synaptic vesicle proteins to clathrin coat proteins that have been fractionated by SDS/PAGE and transferred to nitrocellulose, 2) identifying soluble cytoplasmic factors that bind to the cytoplasmic regions of synaptic vesicle proteins that have been immobilized to a solid matrix. During phase Il, we will determine the tissue specificity of identified sortases, purify and sequence them, and test their involvement in synaptic vesicle biogenesis using an in-vitro reconstitution assay. Plans will also be made for instituting research related to gastrointestinal development, function, and disease.